Amino acid transporters involved in luminal transport of mercuric conjugates of cysteine in rabbit proximal tubule.

The primary aim of the present study was to test the hypothesis that amino acid transport systems are involved in absorptive transport of dicysteinylmercury (cysteine-Hg-cysteine). Luminal disappearance flux [JD, fmol x min(-1) (mm tubular length)(-1)] of inorganic mercury (Hg2+), in the form of dicysteinylmercury, was measured in isolated perfused S2 segments with various amino acids or amino acid analogs in the luminal compartment under one of two conditions, in the presence or absence of Na+. The control perfusion fluid contained 20 microM dicysteinylmercury. Replacing Na+ in both the bathing and perfusing solutions with N-methyl-D-glucamine reduced the JD of Hg2+ by about 40%. Nine amino acids and two amino acid analogs were coperfused individually (at millimolar concentrations) with dicysteinylmercury. The amino acids and amino acid analogs that had the greatest effect on the JD of Hg2+ were L-cystine, L-serine, L-histidine, L-tryptophan, and 2-(-)-endoamino-bicycloheptane-2-carboxylic acid. The greatest reduction (76%) in the total JD of Hg2+ occurred when L-cystine was coperfused with dicysteinylmercury in the presence of Na+. Overall, the current findings indicate that Hg2+ is transported from the lumen into proximal tubular epithelial cells via amino acid transporters that recognize dicysteinylmercury. In addition, the data indicate that multiple amino acid transporters are involved in the luminal uptake of dicysteinylmercury, including the Na+-dependent low-affinity L-cystine, B(0), and ASC systems and the Na+-independent L-system. Furthermore, the transport data obtained when L-cystine was added to the luminal fluid indicate strongly that dicysteinylmercury is likely transported as a molecular homolog of L-cystine.

Some health physics aspects of working with 203Hg in university research.

The radioisotope 203Hg is used in university toxicology research experiments. When our commercial vendor ceased the production of the high specific activity 203Hg we required, an alternative source was sought. Other commercial sources were investigated without success leaving the synthesis of this radioisotope to us. This paper outlines the method we used to synthesize 203Hg and provides a summary of our results to date and a discussion of our experiences.

Disposition of inorganic mercury following biliary obstruction and chemically induced glutathione depletion: dispositional changes one hour after the intravenous administration of mercuric chloride.

Influences of biliary obstruction and systemic depletion of glutathione (GSH) on the disposition of a low nontoxic iv dose of inorganic mercury were evaluated in rats in the present study. Specifically, the disposition of mercury in the kidneys, liver, small and large intestines, and blood was assessed 1 h after the injection of 0.5 micromol/kg mercuric chloride in control rats and rats pretreated with acivicin, buthionine sulfoximine (BSO), or diethylmaleate (DEM) that did or did not undergo acute biliary ligation prior to the injection of mercury. Among the groups that did not undergo biliary ligation, the pretreatments used to alter GSH status systemically had varying effects on the disposition of inorganic mercury in the kidneys, liver, intestines, and blood. Biliary ligation caused the net renal accumulation of mercury to decrease under all pretreatment conditions. By contrast, biliary ligation caused significant increases in the hepatic burden of mercury in all pretreatment groups except the acivicin-pretreated group. Blood levels of mercury also increased as a result of biliary ligation, regardless of the type of pretreatment used. Evidence for a secretory-like movement of mercury into the lumen of the intestines is also provided in the animals that underwent biliary ligation. The present findings indicate that biliary ligation combined with methods used to alter GSH status systemically have additive effects with respect to causing reductions in the net renal accumulation of mercury. In addition, the findings indicate that at least some fraction of the renal accumulation of inorganic mercury is linked mechanistically to the hepatobiliary system.

Relationships between alterations in glutathione metabolism and the disposition of inorganic mercury in rats: effects of biliary ligation and chemically induced modulation of glutathione status.

Influences of biliary ligation and systemic depletion of glutathione (GSH) or modulation of GSH status on the disposition of a low, non-nephrotoxic i.v. dose of inorganic mercury were evaluated in rats in the present study. Renal and hepatic disposition, and the urinary and fecal excretion, of inorganic mercury were assessed 24 h after the injection of a 0.5-micromol/kg dose of mercuric chloride in control rats and rats pretreated with acivicin (two 10-mg/kg i.p. doses in 2 ml/kg normal saline, 90 min apart, 60 min before mercuric chloride), buthionine sulfoximine (BSO; 2 mmol/kg i.v. in 4 ml/kg normal saline, 2 h before mercuric chloride) or diethylmaleate (DEM; 3.37 mmol/kg i.p. in 2 ml/kg corn oil, 2 h before mercuric chloride) that either underwent or did not undergo acute biliary ligation prior to the injection of mercury. Among the groups that did not undergo biliary ligation, the pretreatments used to alter GSH status systemically had varying effects on the disposition of inorganic mercury in the kidneys, liver, and blood. Biliary ligation caused the net renal accumulation of mercury to decrease under all pretreatment conditions. By contrast, biliary ligation caused significant increases in the hepatic burden of mercury in all pretreatment groups except in theacivicin-pretreated group. Blood levels of mercury also increased as a result of biliary ligation, regardless of the type of pretreatment used. The present findings indicate that biliary ligation combined with methods used to modulate GSH status systemically have additive effects with respect to causing reductions in the net renal accumulation of mercury. Additionally, the findings indicate that at least some fraction of the renal accumulation of inorganic mercury is linked mechanistically to the hepato-biliary system.

Mechanisms of action of 2,3-dimercaptopropane-1-sulfonate and the transport, disposition, and toxicity of inorganic mercury in isolated perfused segments of rabbit proximal tubules.

Mechanisms by which the dithiol chelating agent 2, 3-dimercaptopropane-1-sulfonate (DMPS) significantly alters the renal tubular transport, accumulation, and toxicity of inorganic mercury were studied in isolated perfused pars recta (S2) segments of proximal tubules of rabbits. Addition of 200 microM DMPS to the bath provided complete protection from the toxic effects of 20 microM inorganic mercury in the lumen. The protection was linked to decreased uptake and accumulation of mercury. Additional data indicated that, when DMPS and inorganic mercury were coperfused through the lumen, very little inorganic mercury was taken up from the lumen. We also obtained data indicating that DMPS is transported by the organic anion transport system and that this transport is linked to the therapeutic effects of DMPS. Interestingly, very little inorganic mercury was taken up and no cellular pathological changes were detected when inorganic mercury and DMPS were added to the bath. We also tested the hypothesis that DMPS can extract cellular mercury while being transported from the bath into the luminal compartment. Our findings showed that, when DMPS was applied to the basolateral membranes of S2 segments after they had been exposed to mercuric conjugates of glutathione of the laminal membrane, the tubular content of mercury was greatly reduced and the rates of disappearance of mercury from the lumen changed from positive values to markedly negative values. We conclude that inorganic mercury is extracted from proximal tubular cells by a transport process involving the movement of DMPS from the bathing compartment to the luminal compartment.

Heterogeneity of glutathione synthesis and secretion in the proximal tubule of the rabbit.

This study was designed to examine the synthesis and possible secretion of glutathione (GSH) in the S1, S2, and S3 segments of the rabbit proximal tubule. GSH synthesis and secretion rates were measured in the three segments of the proximal tubule, using the isolated perfused renal tubule technique. Tritiated (3H) glycine was perfused into segments and synthesized [3H]GSH (3H on the glycine residue) was measured in the bathing solution, collectate, and tubule extract. In the S1 segments, GSH was synthesized at the rate of 8.65 +/- 0.88 fmol.min-1.mm-1 tubule length and preferentially secreted into the lumen at the rate of 7.28 +/- 0.74 fmol.min-1.mm-1. The difference between synthesis and secretion appeared in the bathing solution. The S2 segment synthesized GSH at the rate of 3.88 +/- 0.82 and secreted GSH at the rate of 2.78 +/- 0.57 fmol.min-1.mm-1. GSH synthesis and secretion rates in the S3 segment were 5.45 +/- 1.19 and 4.22 +/- 1.16 fmol.min-1.mm-1, respectively. Cellular concentrations of [3H]GSH increased along the length of the proximal tubule, with the highest concentrations in the S3 segment. The respective GSH cellular concentrations in the S1, S2, and S3 segments were 35.89 +/- 10.51, 49.65 +/- 9.32, and 116.90 +/- 15.76 microM. These findings indicate that there is heterogeneity of GSH synthesis along the proximal tubule and that synthesized GSH is secreted preferentially into the lumen.

Participation of mercuric conjugates of cysteine, homocysteine, and N-acetylcysteine in mechanisms involved in the renal tubular uptake of inorganic mercury.

Mechanisms involved in the renal uptake of inorganic mercury were studied in rats administered a nontoxic 0.5 mumol/kg intravenous dose of inorganic mercury with or without 2.0 mumol/kg cysteine, homocysteine, or N-acetylcysteine. The renal disposition of mercury was studied 1 h after treatment in normal rats and rats that had undergone bilateral ureteral ligation. In addition, the disposition of mercury (including the urinary and fecal excretion of mercury) was evaluated 24 h after treatment. In normal rats, coadministering inorganic mercury plus cysteine or homocysteine caused a significant increase in the renal uptake of mercury 1 h after treatment. The enhanced renal uptake of mercury was due to increased uptake of mercury in the renal outer stripe of the outer medulla and/or renal cortex. Ureteral ligation caused reductions in the renal uptake of mercury in all groups except for the one treated with inorganic mercury plus N-acetylcysteine. Thus, it appears that virtually all of the mercury taken up by the kidneys of the normal rats treated with inorganic mercury plus N-acetylcysteine occurred at the basolateral membrane. Urinary excretory data also support this notion, in that the rate of excretion of inorganic mercury was greatest in the rats treated with inorganic mercury plus N-acetylcysteine. Our data also indicate that uptake of inorganic mercury in the kidneys of rats treated with inorganic mercury plus cysteine occurred equally at both luminal and basolateral membranes. In addition, the renal uptake of mercury in rats treated with inorganic mercury plus homocysteine occurred predominantly at the basolateral membrane with some component of luminal uptake. The findings of the present study confirm that there are at least two distinct mechanisms involved in the renal uptake of inorganic mercury, with one mechanism located on the luminal membrane and the other located on the basolateral membrane. Our findings also show that cysteine and homologs of cysteine, when coadministered with inorganic mercury, greatly influence the magnitude and/or site of uptake of mercuric ions in the kidney.

Small aliphatic dicarboxylic acids inhibit renal uptake of administered mercury.

We evaluated the effects of pretreating rats intravenously with small aliphatic dicarboxylic acids on the renal disposition of injected inorganic mercury. Three different sets of experiments were carried out. When rats were pretreated with succinic acid, glutaric acid, or adipic acid 5 min prior to the injection of a 0.5-mumol/kg dose of mercuric chloride, there was a significant dose-dependent inhibitory effect on the renal disposition of mercury during the first hour after the administration of mercuric chloride. Both glutaric and adipic acid, at a dose of 1.0 mmol/kg, caused the greatest level of inhibition in the renal tubular uptake of inorganic mercury. By the end of the first hour after the injection of mercuric chloride, the renal burden of mercury in rats pretreated with either glutaric or adipic acid was 27-35% lower than in corresponding control rats. Malonic acid at a dose of 1.0 mmol/kg had no effect on the renal disposition of inorganic mercury. The inhibitory effect of succinic, glutaric, or adipic acid on the overall renal uptake of mercury was due to effects in both the cortex and outer stripe of the outer medulla. Findings from an experiment in which rats had their ureters ligated showed that the inhibitory effect of glutaric acid on the renal tubular uptake of mercury was due to inhibition of the uptake of mercury at the basolateral membrane. Our findings confirm that one of the mechanisms involved in the proximal tubular uptake of inorganic mercury is located on the basolateral membrane. According to findings from our previous studies, this mechanism appears to involve the activity of the organic anion transporter. The inhibitory effects of dicarboxylic acids on the renal tubular uptake of administered inorganic mercury, especially in rats whose ureters had been ligated, are consistent with the hypothesis that the organic anion transport system is involved in the basolateral uptake of inorganic mercury along the proximal tubule.

Nephrotoxicity of inorganic mercury co-administrated with L-cysteine.

In the present study, we tested the hypothesis that co-administration of low nephrotoxic doses of inorganic mercury (Hg++) with L-cysteine (in a 1:2 mol ratio of inorganic mercury to L-cysteine), alters significantly the nephropathy induced by inorganic mercury. In the first experiment, the effect of co-administering L-cysteine on the nephropathy induced by a 1.8 or 2.0 micromol/kg dose of inorganic mercury was evaluated in rats 24 h after the administration of inorganic mercury. According to histopathological assessment of sections of kidney and evaluation of the urinary excretion of lactate dehydrogenase, total protein and inorganic mercury (which were used as indices of renal injury), the severity of renal injury in rats co-administered the L-cysteine with the inorganic mercury was significantly greater than that in corresponding rats injected with only inorganic mercury. In a second experiment, the disposition of mercury was evaluated 1 h after the administration of 1.8 micromol inorganic mercury/kg with or without 3.6 micromol L-cysteine/kg. The renal accumulation of mercury, specifically in the cortex and outer stripe of the outer medulla, was significantly greater the rats co-administered the inorganic mercury and L-cysteine than in the rats given only inorganic mercury. In addition, the content of mercury in the blood and liver was significantly lower, and the fraction of mercury in the blood present in the plasma was significantly greater, in the rats co-administered inorganic mercury and L-cysteine than in the rats given only inorganic mercury. On the basis of the findings from this study, the nephropathy induced by low nephrotoxic doses of inorganic mercury is made more severe when the inorganic mercury is co-administered in a 1:2 mol ratio with L-cysteine. Moreover, it appears that the enhanced severity in the nephropathy induced by the co-administration of inorganic mercury and L-cysteine is linked to an increase in the tubular uptake of mercury in the cortex and outer stripe of the outer medulla.

Diversion or prevention of biliary outflow from the liver diminishes the renal uptake of injected inorganic mercury.

In the present study, we tested the hypothesis that diversion of biliary flow from the liver to the intestines (using biliary cannulation) or prevention of biliary outflow from the liver ( by biliary ligation) affects significantly the renal uptake and accumulation of mercury in rats given an intravenous nontoxic (0.5 mumol/kg) dose of mercuric chloride (containing 203 HgCl2). Diverting biliary flow away from the small intestine, by cannulation of the bile duct, caused a significant increase in the content of mercury in the blood and caused a significant decrease in the total renal uptake of mercury at 1 and 3 hr after the injection of mercuric chloride. By the end of 3 hr after the injection of mercury, the amount of mercury that was not taken up by the kidneys, as a result of diversion of biliary flow, was approximately 10% of the administered dose. The decreased renal uptake of mercury was caused by decreased uptake of mercury in the renal cortex and outer stripe of the outer medulla. Interestingly, very little mercury was excreted in the bile. Only approximately 0.19% of the administered dose of mercury was excreted in the bile in 3 hr. Renal accumulation of mercury, particularly in the cortex and outer stripe of the outer medulla, was also reduced significantly after biliary ligation, when evaluated 24 hr after the injection of inorganic mercury. There was an almost 3-fold increase in the content of mercury in the liver of the rats whose bile duct had been ligated. Fecal excretion of mercury was also diminished in these animals. It was interesting, however, that these rats did excrete some mercury in the feces. Dispositional data obtained from the segments of the gastrointestinal tract indicate that fecal excretion of mercury in the rats whose bile duct had been ligated was most likely caused by intestinal secretion of mercury. In conclusion, the present findings indicate that a hepato-biliary-enteric metabolic pathway plays a role in some aspect of the renal accumulation of administered inorganic mercury. This role does not, however, seem to involve, to any significant degree, the biliary and enteric processing of mercury secreted into the bile.

Pretreatment with p-aminohippurate inhibits the renal uptake and accumulation of injected inorganic mercury in the rat.

The effects of intravenous pretreatment with the organic anion p-aminohippurate (PAH) on the disposition of intravenously administered inorganic mercury in the kidneys, liver and blood were evaluated in rats. In dose-response experiments, the renal uptake (and/or accumulation) of mercury, 1 h after the injection of a nontoxic 0.5 mumol/kg dose of mercuric chloride (HgCl2), was significantly reduced in rats when a 1.0, 3.3 or 10 mmol/kg dose of PAH was administered 5 min prior to the injection of HgCl2. This reduction was due to reduced uptake of mercury in both the renal cortex and outer stripe of the outer medulla. Near maximal inhibition appeared to be achieved with the 10 mmol/kg dose of PAH. Inhibition of the uptake (an/or accumulation) of mercury in the renal cortex and outer stripe of the outer medulla, 1 h after the injection of the nontoxic dose of HgCl2, was also detected in experiments where HgCl2 was injected 5, 30, 60 or 180 min after pretreatment with a 10 mmol/kg dose of PAH. The renal uptake of mercury was inhibited significantly when the nontoxic dose of inorganic mercury was administered 5, 30, or 60, but not 180 min after pretreatment with the 10 mmol/kg dose of PAH. In another experiment, the renal burden of mercury was significantly reduced for 24 h when pretreatment with a 10 mmol/kg dose of PAH was administered 5 min prior to the injection of HgCl2. Pretreatment with PAH did not have an effect on the hepatic disposition of mercury, but it did cause a significant increase in the fraction of mercury present in the plasma of blood. In summary, the findings in the present study indicate that pretreatment with PAH inhibits the renal uptake of injected inorganic mercury in a dose-dependent and time-dependent manner. In addition, the findings tend to indicate that some fraction of the mercury that enters into renal tubular epithelial cells is by a mechanism involving the organic anion transport system.

Accumulation and handling of inorganic mercury in the kidney after coadministration with glutathione.

The accumulation and handling of mercury in the blood, kidneys, and liver were evaluated and compared in rats 5 min, 1 h, and 24 h after the intravenous administration of either a 0.25 mumol/kg dose of inorganic mercury or a 0.25 mumol/kg dose of inorganic mercury plus a 0.5 mumol/kg dose of glutathione (GSH) to determine the possible role of extracellular GSH and complexes of GSH and inorganic mercury in the renal uptake and transport of inorganic mercury. Significantly more of the injected dose of inorganic mercury was present in the blood of the rats injected with inorganic mercury alone than in the blood of the rats injected simultaneously with both inorganic mercury and GSH at all times evaluated after injection. Of the mercury remaining in the blood, however, significantly more mercury was in plasma fraction of blood in the rats injected with both inorganic mercury and GSH than in the plasma fraction of blood in the rats injected with inorganic mercury alone. The blood and plasma findings indicate that much of the mercury injected with GSH was in some complex that allowed the mercury to be cleared from the blood more readily and prevented the mercury from entering readily into red blood cells. The renal concentration of mercury was significantly greater in the rats injected with both inorganic mercury and GSH than in the rats injected with inorganic mercury alone at 5 min and 1 h, but not 24 h, after injection. This increased renal accumulation of mercury during the initial hours after injection was due mainly to enhanced uptake and/or retention of mercury in the renal cortex. Urinary excretion of mercury, over 24 h, was also slightly, but significantly, greater in the rats injected with both inorganic mercury and GSH simultaneously. These data indicate that coadministration of a nontoxic dose of inorganic mercury with a twofold higher amount (in moles) of GSH increases significantly the clearance of mercury from the blood and increases the renal cortical accumulation of inorganic mercury during the initial 1 h after injection. Moreover, the data in this study are consistent with the hypothesis that extracellular GSH is an important ligand to which mercuric ions bind, and that complexes of inorganic mercury and GSH in the blood and/or ultrafiltrate probably play a role in the renal uptake of some of the mercury in blood after exposure to mercuric compounds.

Renal disposition of mercury in rats after intravenous injection of inorganic mercury and cysteine.

The disposition of mercury in the blood, kidneys and liver was evaluated and compared in rats 5 min, 1 h, and 24 h after the intravenous administration of a 0.25 mumol/kg dose of inorganic mercury or a 0.25 mumol/kg dose of inorganic mercury plus a 0.5 mumol/kg dose of cysteine to determine the possible role of extracellular cysteine and complexes of cysteine and inorganic mercury in the renal uptake and transport of inorganic mercury. More inorganic mercury was present in the blood of the rats injected with inorganic mercury alone than in the blood of the rats injected simultaneously with both the inorganic mercury and cysteine during the first hour after injection. In addition, significantly more mercury was in the plasma fraction of blood in the rats injected with both inorganic mercury and cysteine than in the rats injected with inorganic mercury alone. These findings indicate that much of the mercury injected with cysteine was in some form of a complex that allowed the mercury to be cleared from the blood more readily and prevented the mercury from entering readily into the cellular components of blood. The renal concentration of mercury was significantly greater in the rats injected with both inorganic mercury and cysteine than in the rats injected with inorganic mercury alone 1 h, but not 24 h, after injection. This increased renal accumulation of mercury during the initial hour after injection was due mainly to enhanced uptake and/or retention of mercury in the renal cortex, although some of the enhanced accumulation of mercury also occurred in the outer stripe of the outer medulla during the first hour after injection. These data indicate that coadministration of a nontoxic dose of inorganic mercury with a twofold higher amount (in moles) of cysteine increases significantly the clearance of mercury from the blood and increases the accumulation of inorganic mercury in the renal cortex and outer stripe of the outer medulla during the initial 1 h after injection. In conclusion, the data in this study are consistent with the hypothesis that complexes of inorganic mercury and cysteine in the blood and/or ultrafiltrate probably play a role in the renal uptake of some of the mercury in blood after exposure to mercuric compounds.

Lack of luminal or basolateral uptake and transepithelial transport of mercury in isolated perfused proximal tubules exposed to mercury-metallothionein.

The lumen-to-bath and bath-to-lumen transport, cellular uptake, and toxicity of inorganic mercury bound to metallothionein (203Hg-MT) were studied in isolated perfused S1, S2, and S3 segments of the renal proximal tubule of rabbits. Evidence of very mild toxicity was displayed in some of the segments perfused through the lumen with 18.4 microM inorganic mercury in the form of Hg-MT. The toxic response was restricted primarily to mild swelling of the epithelial cells localized at the end of the tubular segments where the perfusion pipette was inserted into the lumen. The cells in the proximal portions of perfused S2 segments appeared to be most severely affected in that a few blebs would on occasion come off the epithelial cells. Mild cellular swelling was also observed in some S2 and S3 segments that were exposed to 18.4 microM inorganic mercury in the form of Hg-MT in the bath. The swelling was more generalized, involving all the epithelial cells along the perfused segment. Very little, or no, measurable lumen-to-bath or bath-to-lumen transport of Hg as Hg-MT could be detected in any of the 3 perfused segments of the proximal tubule during 40-45 min of perfusion. The complex of Hg-MT appeared to behave in a manner similar to that of the volume marker [3H]-L-glucose. The lack of tubular transport of Hg as Hg-MT was confirmed by little or no measurable uptake and accumulation of inorganic mercury in the tubular epithelial cells. Thus, our findings indicate that the Hg-MT complex is not taken up avidly in isolated perfused S1, S2, or S3 segments of the proximal tubule.

Mercury-metallothionein and the renal accumulation and handling of mercury.

In the present study, we evaluated the renal and hepatic accumulation of mercury, the intrarenal distribution of mercury and the urinary and fecal excretion of mercury in rats injected intravenously with a non-toxic 0.1 mumol/kg-dose of mercury in the form of mercuric chloride (HgCl2) or a complex of mercury-metallothionein (Hg-MT). Between 6 and 72 h after injection, the concentration of mercury in the kidneys of the rats injected with Hg-MT was significantly greater than that in the rats injected with HgCl2. The greatest difference in the renal concentration of mercury between the two groups of rats was detected 6 h after injection. In the kidneys of both experimental groups of rats, the cortex and the outer stripe of the outer medulla contained the highest concentrations of mercury, with the greatest concentrations found in the renal cortex and outer stripe of the outer medulla of the rats injected with Hg-MT. No differences were found between the two experimental groups with respect to the concentration of mercury in the renal inner stripe of the outer medulla and inner medulla throughout 72 h of study. The content of mercury in the blood and liver decreased over time in both groups of rats, but was always significantly greater in the blood and liver of rats injected with HgCl2. The rats injected with Hg-MT excreted more than eight times the amount of mercury in the urine than the corresponding rats injected with HgCl2 during 72 h. These data indicate that there may be decreased tubular reabsorption of filtered Hg-MT and/or tubular secretion of mercury in the rats injected with Hg-MT. In contrast, the rats injected with HgCl2 excreted significantly more mercury in the feces during the same period of time than the corresponding rats injected with Hg-MT. In conclusion, our data clearly indicate that the renal and hepatic uptake and accumulation of mercury, and the urinary and fecal excretion of mercury, are altered significantly when inorganic mercury is administered intravenously as a complex with metallothionein.

Intrarenal distribution of inorganic mercury and albumin after coadministration.

The renal disposition and the intrarenal distribution of albumin and mercury were studied simultaneously in rats co-injected with a 0.5-mumol/kg dose of albumin and a 0.25-mumol/kg dose of inorganic mercury at 2, 5, 30, and 180 min after injection. These studies were carried out to test the hypothesis that one of the mechanisms involved in the renal tubular uptake of inorganic mercury is cotransport with albumin. By the end of the first 2 min after injection, the ratio of inorganic mercury to albumin in the renal cortex and outer stripe of the outer medulla was approximately 2.6 and 1.6, respectively. Both the cortex and outer stripe contain segments of the proximal tubule, and it is these segments that have been shown to be principally involved in the renal tubular uptake of both albumin and inorganic mercury. The ratio increased slightly in these two zones after 5 and 20 min after injection. These data demonstrate that there is a relatively close relationship in the renal content of inorganic mercury and albumin during the early minutes after coinjection of inorganic mercury and albumin. However, the ratios are significantly greater than the ratio of inorganic mercury to albumin in the injection solution, which was 0.5. After 180 min following co-injection, the ratio increased to about 38 in the cortex and 15 in the outer stripe. This increase in the ratio is probably related to the metabolism of albumin. Based on the ratios of inorganic mercury to albumin in the renal cortex and outer stripe of the outer medulla, it appears that some proximal tubular uptake of inorganic mercury occurs by mechanisms other than endocytotic cotransport of inorganic mercury with albumin. However, since the ratios were small during the early times after injection, cotransport of inorganic mercury with albumin cannot be excluded as one of the mechanisms involved in the proximal tubular uptake of inorganic mercury.

Transport and toxicity of methylmercury along the proximal tubule of the rabbit.

Toxicity and transport of methylmercury were studied in isolated perfused S1, S2, and S3 segments of the renal proximal tubule of the rabbit. Methylmercury (II) chloride, ranging from 1 nM-1 mM, was perfused through the lumen of the three segments for up to 60 min. Lumen-to-bath transport of methylmercury was studied when the concentration of methylmercury in the perfusing solution was 18.4 microM. S1 segments of the proximal tubule were most vulnerable to the toxic effects of methylmercury. Cellular swelling and blebbing occurred when the concentration of methylmercury in the perfusate was as low as 1 nM. In the S2 and S3 segments, morphologically discernable cellular injury did not occur until the concentration of methylmercury in the perfusate was greater than 100 nM. Due to severe cellular injury and luminal occlusion, transport data could not be obtained from S1 segments. However, transport could be measured in both S2 and S3 segments. Methylmercury (18.4 microM) disappeared from the luminal fluid across the luminal membrane (JD) very rapidly in both segments. The rate was so rapid that about 80% of the methylmercury that entered the luminal fluid was abstracted. Interestingly, the rate at which mercury appeared in the bathing solution (JA) was statistically equivalent to the JD. Since the predicted leak of methylmercury was very low, most of the JA represented actual transepithelial flux of methylmercury. When 80 microM glutathione (GSH) was added to the perfusate along with 18.4 microM methylmercury, the JD was decreased significantly in both S2 and S3 segments. Moreover, the addition of 80 microM GSH caused cellular injury to be exacerbated in the S3 segments. In conclusion, there are differences in the toxicity of methylmercury along the three segments of the proximal tubule when the methylmercury is delivered to the lumen of the segments in a simple electrolyte solution. Methylmercury is very avidly transported across the tubular epithelium in S2 and S3 segments of the proximal tubule. In addition, when 80 microM GSH is added to the perfusate, the toxicity and transport of methylmercury are modified.

Cadmium transport and toxicity in isolated perfused segments of the renal proximal tubule.

We measured the lumen-to-bath transport and assessed the toxicity of inorganic cadmium (Cd2+) in isolated, perfused segments of the rabbit renal proximal tubule. To determine the dose range for acute toxicity the segments (S1, S2, and S3) were perfused with cadmium chloride (CdCl2) and the vital dye, FD & C green. We observed the tubular epithelial cells under the light microscope for signs of cellular injury and necrosis. Cellular swelling, blebbing of the luminal membrane, and cellular vacuolization were indicators of cellular injury, and the uptake of dye was indicative of cellular necrosis. Visible cellular damage occurs within 45 min after exposure of renal proximal tubular cells to cadmium concentrations greater than 500 microM. To determine rates of transport and cellular uptake of cadmium, the segments were perfused with a mixture of 109CdCl2 and the volume marker, L-[3H]glucose. We added nonradioactive CdCl2 to vary the total cadmium concentration from 1.5 to 2000 microM. After perfusion, we treated the tubules with 3% trichloroacetic acid or with a buffer solution of reduced osmolality in an attempt to determine the fate of the cadmium reabsorbed from the lumen. The tubular transport of cadmium was measured as the rate of disappearance of cadmium from the lumen (JD, pmol min-1 mm-1) and as the rate of appearance of cadmium in the bath (JA, pmol min-1 mm-1). In transport experiments, increasing the concentration of cadmium in the lumen caused an increase in the leak of the volume marker from the lumen into the bath. Cadmium disappeared from the lumen much more rapidly than it appeared in the bath for all three tubular segments. We conclude that (i) ionic cadmium, at concentrations greater than 500 microM, is acutely toxic to cells of isolated, perfused renal proximal tubules, and this toxicity is greater in the S1 than in the S2 or S3 segments; (ii) it is avidly taken up at the luminal membrane in all three segments; uptake is greater in the S1 than in the S2 or S3 segments; (iii) less than 10% of the cadmium that disappears from the lumen is transported across the basolateral membrane into the bath; and (iv) appearance flux into the bath does not show saturation in any of the segments over the concentration range studied; disappearance flux from the lumen shows saturation in the S2 and S3 segments, but not in the S1 segment.

Altered intrarenal accumulation of mercury in uninephrectomized rats treated with methylmercury chloride.

We tested the hypothesis that the intrarenal accumulation of mercury in rats treated with methylmercury is altered significantly as a result of unilateral nephrectomy and compensatory renal growth. Renal accumulation of mercury was evaluated by radioisotopic techniques in both uninephrectomized (NPX) and sham-operated (SO) rats 1, 2, and 7 days after the animals received a nonnephrotoxic intravenous dose of methylmercury chloride (5 mg/kg Hg). At all times studied after the injection of the dose of methylmercury, the renal accumulation of mercury (on a per gram kidney basis) was significantly greater in the NPX rats than that in the SO rats. The increased accumulation was due to a specific increase in the accumulation of mercury in the outer stripe of the outer medulla. Renal cortical accumulation of mercury was similar in both the NPX and SO rats. The percentage of the administered dose of mercury that was present in the total renal mass of the NPX and SO rats ranged between 5 and 15, depending on the day that the renal accumulation was studied. Approximately 40-50% of the total renal burden of mercury in both the NPX and SO rats was in the inorganic form. However, only less than 1% of the mercury in blood was in the inorganic form at the three times accumulation was studied. Very little mercury was excreted in the urine by either the NPX or SO rats. Only about 2 to 3% of the administered dose of mercury was excreted in the urine in 7 days. By contrast, the cumulative fecal excretion of mercury over 7 days was substantial in the NPX and SO rats, and significantly more mercury was excreted in the feces by the NPX rats (about 19% of the dose) than by that in the SO rats (about 16% of the dose). In conclusion, our findings indicate that unilateral nephrectomy and compensatory renal growth cause a significant increase in the accumulation of mercury in the renal outer stripe of the outer medulla in rats exposed to methylmercury. In addition, the findings indicate that the fecal excretion of mercury is also significantly increased.

Axial heterogeneity of adenosine transport and metabolism in the rabbit proximal tubule.

Transport and metabolism of adenosine were studied in the S1, S2, and S3 segments of the rabbit proximal renal tubule. Isolated segments were perfused in vitro with uniformly labelled 14C-adenosine to measure the lumen-to-bath flux of adenosine. This flux rate was measured by the disappearance of 14C from the luminal fluid (JD) and simultaneously by the appearance of 14C in the bathing solution (JA), expressed as femtomoles per minute per millimeter of tubule length (fmol.min-1.mm-1). At a perfused concentration of 83.3 microM adenosine, when corrected for metabolism, the JDs for adenosine in the S1, S2, and S3 segments were 735, 212, and 273, respectively. JAs, corrected for metabolism, were 0, 0, and 4.8 fmol.min-1.mm-1 for the S1, S2, and S3 segments, indicating that very little or no 14C-adenosine moved across the basolateral membrane. To correct for metabolism of 14C-adenosine, the perfusion fluid, collected fluid, tubular extract, and bathing fluid, from three tubules of each segment type, were analyzed by high-performance liquid chromatography to identify 14C-adenosine and its 14C-metabolites. At 83.3 microM, all segments metabolized adenosine extensively. Consequently, adenosine-5'-monophosphate (AMP) and inosine were found in tubule cells of all segments. Inosine also appeared in the collected fluid, but AMP did not. In S1 and S2 segments, none of the 14C in the bathing solutions could be identified and no adenosine was found. Of the small amounts of 14C found in bathing solutions from S3 segments, about 27% appeared to be adenosine, the rest were inosine and hypoxanthine or unidentified metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)